2alpha-Methyl and 2beta-methyl analogs of 19,26,27-trinor-(20S)-1alpha-hydroxyvitamin D3 and their uses

ABSTRACT

This invention discloses 2α-methyl and 2β-methyl analogs of 19,26,27-trinor-(20S) -1α-hydroxyvitamin D 3  and pharmaceutical uses therefor. These compounds exhibit pronounced activity in arresting the proliferation of undifferentiated cells and inducing their differentiation to the monocyte thus evidencing use as an anti-cancer agent and for the treatment of skin diseases such as psoriasis as well as skin conditions such as wrinkles, slack skin, dry skin and insufficient sebum secretion. These compounds also have little, if any, calcemic activity and therefore may be used to treat autoimmune disorders or inflammatory diseases in humans as well as renal osteodystrophy. These compounds may also be used for the treatment or prevention of obesity.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.60/630,183, filed Nov. 22, 2004.

BACKGROUND OF THE INVENTION

This invention relates to vitamin D compounds, and more particularly to2α-methyl and 2β-methyl analogs of19,26,27-trinor-(20S)-1α-Hydroxyvitamin D₃ and their pharmaceuticaluses.

The natural hormone, 1α,25-dihydroxyvitamin D₃ and its analog inergosterol series, i.e. 1α,25-dihydroxyvitamin D₂ are known to be highlypotent regulators of calcium homeostasis in animals and humans, andtheir activity in cellular differentiation has also been established,Ostrem et al., Proc. Natl. Acad. Sci. USA, 84, 2610 (1987). Manystructural analogs of these metabolites have been prepared and tested,including 1α-hydroxyvitamin D₃, 1α-hydroxyvitamin D₂, various side chainhomologated vitamins and fluorinated analogs. Some of these compoundsexhibit an interesting separation of activities in cell differentiationand calcium regulation. This difference in activity may be useful in thetreatment of a variety of diseases such as renal osteodystrophy, vitaminD-resistant rickets, osteoporosis, psoriasis, and certain malignancies.

Another class of vitamin D analogs, i.e. the so called 19-nor-vitamin Dcompounds, is characterized by the replacement of the A-ring exocyclicmethylene group (carbon 19), typical of the vitamin D system, by twohydrogen atoms. Biological testing of such 19-nor-analogs (e.g.,1α,25-dihydroxy-19-nor-vitamin D₃) revealed a selective activity profilewith high potency in inducing cellular differentiation, and very lowcalcium mobilizing activity. Thus, these compounds are potentiallyuseful as therapeutic agents for the treatment of malignancies, or thetreatment of various skin disorders. Two different methods of synthesisof such 19-nor-vitamin D analogs have been described (Perlman et al.,Tetrahedron Lett. 31, 1823 (1990); Perlman et al., Tetrahedron Lett. 32,7663 (1991), and DeLuca et al., U.S. Pat. No. 5,086,191).

In U.S. Pat. No. 4,666,634, 2β-hydroxy and alkoxy (e.g., ED-71) analogsof 1α,25-dihydroxyvitamin D₃ have been described and examined by Chugaigroup as potential drugs for osteoporosis and as antitumor agents. Seealso Okano et al., Biochem. Biophys. Res. Commun. 163, 1444 (1989).Other 2-substituted (with hydroxyalkyl, e.g., ED-120, and fluoroalkylgroups) A-ring analogs of 1α,25-dihydroxyvitamin D₃ have also beenprepared and tested (Miyamoto et al., Chem. Pharm. Bull. 41, 1111(1993); Nishii et al., Osteoporosis Int. Suppl. 1, 190 (1993); Posner etal., J. Org. Chem. 59, 7855 (1994), and J. Org. Chem. 60, 4617 (1995)).

2-Substituted analogs of 1α,25-dihydroxy-19-nor-vitamin D₃ have alsobeen synthesized, i.e. compounds substituted at 2-position with hydroxyor alkoxy groups (DeLuca et al., U.S. Pat. No. 5,536,713), with 2-alkylgroups (DeLuca et al U.S. Pat. No. 5,945,410), and with 2-alkylidenegroups (DeLuca et al U.S. Pat. No. 5,843,928), which exhibit interestingand selective activity profiles. All these studies indicate that bindingsites in vitamin D receptors can accommodate different substituents atC-2 in the synthesized vitamin D analogs.

In a continuing effort to explore the 19-nor class of pharmacologicallyimportant vitamin D compounds, analogs which are characterized by thepresence of a methylene substituent at carbon 2 (C-2), a hydroxyl groupat carbon 1 (C-1), and a shortened side chain attached to carbon 20(C-20) have also been synthesized and tested.1α-Hydroxy-2-methylene-19-nor-pregnacalciferol is described in U.S. Pat.6,566,352 while 1α-hydroxy-2-methylene-19-nor-homopregnacalciferol isdescribed in U.S. Pat. 6,579,861 and1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol is described inU.S. Pat. 6,627,622. All three of these compounds have relatively highbinding activity to vitamin D receptors and relatively high celldifferentiation activity, but little if any calcemic activity ascompared to 1α,25-dihydroxyvitamin D₃. Their biological activities makethese compounds excellent candidates for a variety of pharmaceuticaluses, as set forth in the '352, '861 and '622 patents.

SUMMARY OF THE INVENTION

The present invention is directed toward 2α-methyl and 2β-methyl analogsof 19, 26,27-trinor-(20S)-vitamin D₃, and particularly to the 2α-methyland 2β-methyl analogs of 19,26,27-trinor-(20S)-1α-hydroxyvitamin D₃,their biological activity, and various pharmaceutical uses for thesecompounds.

Structurally the 2α-methyl and 2β-methyl trinor vitamin D₃ analogs arecharacterized by the general formula I shown below:

were the methyl group attached to carbon 2 of the A-ring is in eitherthe R or S configuration, as indicated by the wavy line in the aboveformula I, and where each of X₁ and X₂, which may be the same ordifferent, is selected from hydrogen or a hydroxy-protecting group. Thepreferred analogs are 2α-methyl-19,26,27-trinor-(20S)-1α-hydroxyvitaminD₃ which has the formula Ia:

and 2β-methyl-19,26,27-trinor-(20S)-1α-hydroxyvitamin D₃, which has theformula Ib

The above compounds of formula I exhibit a desired, and highlyadvantageous, pattern of biological activity. The 2α-methyl analog Ia ischaracterized by relatively high binding to vitamin D receptors, but ithas very low ability to mobilize calcium from bone, as compared to1α,25-dihydroxyvitamin D₃. The 2β-methyl analog Ib is characterized byrelatively low binding to vitamin D receptors, and as such would beconsidered relatively inactive in this regard. Hence, these twocompounds can be characterized as having little, if any, calcemicactivity. It is undesirable to raise serum calcium to supraphysiologiclevels when suppressing the preproparathyroid hormone gene (Darwish &DeLuca, Arch. Biochem. Biophys. 365, 123-130, 1999) and parathyroidgland proliferation. These analogs having little or no calcemic activitywhile very active on differentiation are expected to be useful as atherapy for suppression of secondary hyperparathyroidism of renalosteodystrophy.

The compounds of formula I of this invention have also been discoveredto be especially suited for treatment and prophylaxis of human disorderswhich are characterized by an imbalance in the immune system, e.g. inautoimmune diseases, including multiple sclerosis, lupus, diabetesmellitus, host versus graft rejection, and rejection of organtransplants; and additionally for the treatment of inflammatorydiseases, such as rheumatoid arthritis, asthma, and inflammatory boweldiseases such as celiac disease, ulcerative colitis and Crohn's disease.Acne, alopecia and hypertension are other conditions which may betreated with the compound of the invention.

The above compounds of formula I are also characterized by relativelyhigh cell differentiation activity. Thus, these compounds also provide atherapeutic agent for the treatment of psoriasis, or as an anti-canceragent, especially against leukemia, colon cancer, breast cancer, skincancer and prostate cancer. In addition, due to their relatively highcell differentiation activity, these compounds provide therapeuticagents for the treatment of various skin conditions including wrinkles,lack of adequate dermal hydration, i.e. dry skin, lack of adequate skinfirmness, i.e. slack skin, and insufficient sebum secretion. Use ofthese compounds thus not only results in moisturizing of skin but alsoimproves the barrier function of skin.

The compounds of the invention of formula I, and particularly formulaIa, are also useful in preventing or treating obesity, inhibitingadipocyte differentiation, inhibiting SCD-1 gene transcription, and/orreducing body fat in animal subjects. Therefore, in some embodiments, amethod of preventing or treating obesity, inhibiting adipocytedifferentiation, inhibiting SCD-1 gene transcription, and/or reducingbody fat in an animal subject includes administering to the animalsubject, an effective amount of one or more of the compounds or apharmaceutical composition that includes one or more of the compounds offormula I. Administration of one or more of the compounds or thepharmaceutical compositions to the subject inhibits adipocytedifferentiation, inhibits gene transcription, and/or reduces body fat inthe animal subject.

One or more of the compounds may be present in a composition to treatthe above-noted diseases and disorders in an amount from about 0.0 μg/gmto about 1000 μg/gm of the composition, preferably from about 0.1 μg/gmto about 500 μg/gm of the composition, and may be administeredtopically, transdermally, orally, rectally, nasally, sublingually orparenterally in dosages of from about 0.01 μg/day to about 1000 μg/day,preferably from about 0.1 μg/day to about 500 μg/day.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-4 illustrate various biological activities of the 2α-methyl and20β-methyl analogs of 19,26,27-trinor-(20S)-1α-hydroxyvitamin D₃,hereinafter referred to as “C-19”(2α-methyl analog) and “N-16”(2β-methylanalog), respectively, as compared to the native hormone1α,25-dihydroxyvitamin D₃, hereinafter “1,25(OH)₂D₃.”

FIG. 1 is a graph illustrating the relative activity of C-19, N-16 and1,25(OH)₂D₃ to compete for binding with [³H]-1,25-(OH)₂-D₃ to thefull-length recombinant rat vitamin D receptor;

FIG. 2 is a graph illustrating the percent HL-60 cell differentiation asa function of the concentration of C-19, N-16 and 1,25(OH)₂D₃;

FIG. 3 is a bar graph illustrating the in vitro transcription activityof 1,25(OH)₂D₃ as compared to C-19 and N-16; and

FIG. 4 is a bar graph illustrating the change is serum calcium frombaseline of 1,25(OH)₂D₃ as compared to C-19.

DETAILED DESCRIPTION OF THE INVENTION

The 2α-methyl and 2β-methyl analogs of19,26,27-trinor-(20S)-1α-hydroxyvitamin D₃ (referred to herein as C-19and N-16, respectively) were synthesized and tested. Structurally, these2α-methyl and 2β-methyl 19-nor analogs are characterized by the generalformulae Ia and Ib respectively previously illustrated herein, and theirpro-drugs (in protected hydroxy forms) are also illustrated by generalformula I.

The preparation of the 2α-methyl and 2β-methyl analogs of19,26,27-trinor-(20S) -vitamin D₃ having the structure I, Ia and Ib canbe accomplished by a common general method, i.e. the condensation of abicyclic Windaus-Grundmann type ketone II with the allylic phosphineoxide III to the corresponding 2-methylene-19,26,27-trinor-vitamin Danalog IV followed by deprotection at C-1 and C-3 in the lattercompound, and finally conversion of the 2-methylene group in IV to amixture of the 2α-methyl and 2β-methyl compounds of structures Ia and Ibwhich can then be readily separated to provide both epimers (C-19 andN-16).

In the structures III and IV, groups X₁ and X₂ are hydroxy-protectinggroups, preferably t-butyldimethylsilyl. The process shown aboverepresents an application of the convergent synthesis concept, which hasbeen applied effectively for the preparation of vitamin D compounds[e.g. Lythgoe et al., J. Chem. Soc. Perkin Trans. I, 590 (1978);Lythgoe, Chem. Soc. Rev. 9, 449 (1983); Toh et al., J. Org. Chem. 48,1414 (1983); Baggiolini et al., J. Org. Chem. 51, 3098 (1986); Sardinaet al., J. Org. Chem. 51, 1264 (1986); J. Org. Chem. 51, 1269 (1986);DeLuca et al., U.S. Pat. No. 5,086,191; DeLuca et al., U.S. Pat. No.5,536,713].

The hydrindanone of the general structure II is not known. It can beprepared by the method shown on Schemes 1 and 2 (see the preparation ofcompounds C-19 and N-16).

For the preparation of the required phosphine oxides of generalstructure III, a synthetic route has been developed starting from amethyl quinicate derivative which is easily obtained from commercial(1R,3R,4S,5R)-(-)-quinic acid as described by Sicinski et al., J. Med.Chem. 41, 4662 (1998), and by DeLuca and Sicinski, U.S. Pat. No.5,843,928.

The overall process of the synthesis of compounds I, Ia and Ib isillustrated and described more completely in U.S. Pat. No. 5,945,410entitled “2-Alkyl-19-Nor-Vitamin D Compounds” the specification of whichis specifically incorporated herein by reference.

As used in the description and in the claims, the term“hydroxy-protecting group” signifies any group commonly used for thetemporary protection of hydroxy functions, such as for example,alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl groups (hereinafterreferred to simply as “silyl” groups), and alkoxyalkyl groups.Alkoxycarbonyl protecting groups are alkyl—O—CO- groupings such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl,butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl,benzyloxycarbonyl or allyloxycarbonyl. The term “acyl” signifies analkanoyl group of 1 to 6 carbons, in all of its isomeric forms, or acarboxyalkanoyl group of 1 to 6 carbons, such as an oxalyl, malonyl,succinyl, glutaryl group, or an aromatic acyl group such as benzoyl, ora halo, nitro or alkyl substituted benzoyl group. The word “alkyl” asused in the description or the claims, denotes a straight-chain orbranched alkyl radical of 1 to 10 carbons, in all its isomeric forms.Alkoxyalkyl protecting groups are groupings such as methoxymethyl,ethoxymethyl, methoxyethoxymethyl, or tetrahydrofuranyl andtetrahydropyranyl. Preferred silyl-protecting groups are trimethylsilyl,triethylsilyl, t-butyldimethysilyl, dibutylmethylsilyl,diphenylmethylsilyl, phenyldimethylsilyl, diphenyl-t-butylsilyl andanalogous alkylated silyl radicals. The term “aryl” specifies a phenyl-,or an alkyl-, nitro- or halo-substituted phenyl group.

A “protected hydroxy” group is a hydroxy group derivatised or protectedby any of the above groups commonly used for the temporary or permanentprotection of hydroxy functions, e.g. the silyl, alkoxyalkyl, acyl oralkoxycarbonyl groups, as previously defined. The terms “hydroxyalkyl”,“deuteroalkyl” and “fluoroalkyl” refer to an alkyl radical substitutedby one or more hydroxy, deuterium or fluoro groups respectively.

More specifically, reference should be made to the following descriptionas well as to Schemes 1, 2 and 3 herein for a detailed illustration ofthe preparation of compounds of formula I, and specifically compoundsC-19 and N-16.

Preparation of(20S)-de-A,B-8β-(tert-butyldimethylsilyl)oxy-20-(hydroxymethyl)pregnane(2).

Ozone was passed through a solution of vitamin D₂ (3 g, 7.6 mmol) inmethanol (250 mL) and pyridine (2.44 g, 2.5 mL, 31 mmol) for 50 min at−78° C. The reaction mixture was then flushed with an oxygen for 15 minto remove the residual ozone and the solution was treated with NaBH₄(0.75 g, 20 mmol). After 20 min the second portion of NaBH₄ (0.75 g, 20mmol) was added and the mixture was allowed to warm to room temperature.The third portion of NaBH₄ (0.75 g, 20 mmol) was then added and thereaction mixture was stirred for 18 h. The reaction was quenched withwater (40 mL) and the solution was concentrated under reduced pressure.The residue was extracted with ethyl acetate (3×80 mL) and the combinedorganic phase was washed with 1M aq. HCl, saturated aq. NaHCO₃, dried(Na₂SO₄) and concentrated under reduced pressure. The residue waschromatographed on silica gel with hexane/ethyl acetate (75:25) to give(20S)-de-A,B-20-(hydroxymethyl)pregnan-8β-ol 1 (1.21 g, 75% yield) aswhite crystals.

tert-Butyldimethylsilyl trifluoromethanesulfonate (3.24 mL, 3.72 g, 14.1mmol) was added to a solution of the 8β,20-diol 1 (1 g, 4.7 mmol) and2,6-lutidine (1.64 mL, 1.51 g, 14.1 mmol) in anhydrous DMF (15 mL) at 0°C. The mixture was stirred under argon at 0° C. for 1 h and then at roomtemperature for 18 h. The reaction was quenched with water (50 mL) andextracted with ethyl acetate (3×30 mL). The combined organic phase waswashed with brine, dried (Na₂SO₄) and concentrated under reducedpressure. The residue was dissolved in anhydrous THF (8 mL),triethylamine (3 mL, 2.17 g, 21.5 mmol) and a solution oftetrabutylammonium fluoride (1 M in THF, 6.5 mL, 6.5 mmol) were added,followed by freshly activated molecular sieves 4A (3 g). The reactionmixture was stirred under argon at room temperature for 4 h, thenfiltered through a short layer of Celite and evaporated. The residue wasdissolved in ethyl acetate (30 mL), washed with brine, water, dried(Na₂SO₄) and concentrated under reduced pressure. The pure alcohol 2(1.42 g, 93% yield) was isolated by a chromatography on silica gel withhexanelethyl acetate (97.5:2.5→95:5), as a colorless oil: ¹H NMR (500MHz, CDCl₃) δ4.00 (1H, d, J=2.4 Hz, 8α-H), 3.63 (1H, dd, J=10.5, 3.2 Hz,22-H), 3.39 (1H, dd, J=10.5, 6.8 Hz, 22-H), 1.94(1H, br.d, J=12.5 Hz),1.02 (3H, d, J=6.6 Hz, 21-H₃), 0.924 (3H, s, 18-H₃), 0.882 (9H1, s,Si-t-Bu), 0.005 and −0.010 (each 3H, each s, each Si-Me); ¹³CNMR (125MHz) δ69.29 (d, C-8), 67.94 (t, C-22), 53.06 (d), 52.80 (d), 42.12 (s,C-13), 40.54 (t), 38.27 (d), 34.39 (t), 26.79 (t), 25.79 (q, SiCMe₃),23.08 (t), 18.00 (s, SiCMe₃), 17.61 (t), 16.65 (q, C-21), 13.75 (q,C-18), −4.81 and −5.18 (each q, each SiMe.

Preparation of(20S)-de-A,B-8β-(tert-butyldimethylsilyl)oiy-20-formylpregnane (3).

Sulfur trioxide pyridine complex (1.32 g, 8.28 mmol) was added to asolution of the alcohol 2 (451 mg, 1.38 mmol), triethylamine (960 μL,697 mg, 6.9 mmol) in anhydrous methylene chloride (20 mL) and anhydrousDMSO (5 mL) at 0° C. The reaction mixture was stirred under argon at 0°C. for 20 min. and then concentrated. The residue was purified by columnchromatography on silica gel with hexane/ethyl acetate (95:5) to givethe aldehyde 3 (364 mg, 81% yield) as an oil: ¹H NMR (500 MHz, CDCl₃)δ9.55 (1H, d, J=3.1 Hz, CHO), 4.00 (1H, s, 8α-H), 2.33 (1H, m, 20-H),1.89 (1H, dm, J=12.4 Hz), 1.07 (3H, d, J=6.8 Hz, 21-H₃), 0.939 (3H, s,18-H₃), 0.862 (9H, s, Si-t-Bu), −0.009 and −0.026 (each 3H, each s, eachSiMe); ¹³C NMR (125 MHz) δ205.37 (d, CHO), 68.99 (d, C-8), 52.28 (d),51.58 (d), 49.15 (d), 42.58 (s, C-13), 40.35 (t), 34.29 (t), 26.16 (t),25.74 (q, SiCMe₃), 23.27 (t), 17.96 (s, SiCMe₃), 17.52 (t), 14.04 (q,C-21), 13.28 (q, C-18), −4.85 and −5.23 (each q, each SiMe).

Preparation of (20R)-de-A,B8β(tert-butyldimethylsilyl)oxy-20-(hydroxymethyl)pregnane (4).

The aldehyde 3 (364 mg, 1.12 mmol) was dissolved in methylene chloride(15 rnL) and a 40% aq. n-Bu₄NOH solution (1.47 mL, 1.45 g, 2.24 mmol)was added. The resulting mixture was stirred under argon at roomtemperature for 16 h, diluted with methylene chloride (20 mL), washedwith water, dried (Na₂SO₄) and concentrated under reduced pressure. Aresidue was chromatographed on silica gel with hexane/ethyl acetate(95:5) to afford a mixture of aldehyde 3 and its 20-epimer (292 mg, 80%yield) in ca. 1:2 ratio (by ¹H NMR).

This mixture of aldehydes (292 mg, 0.9 mmol) was dissolved in THF (5 mL)and NaBH₄ (64 mg, 1.7 mmol) was added, followed by a dropwise additionof ethanol (5 mnL). The reaction mixture was stirred at room temperaturefor 30 min and it was quenched with a saturated aq. NH₄Cl solution. Themixture was extracted with ether (3×20 mL) and the combined organicphase was washed with water, dried (Na₂SO₄) and concentrated underreduced pressure. The residue was chromatographed on silica gel withhexane/ethyl acetate (96:4→80:20) to give the desired, pure(20R)-alcohol 4 (160 mg, 55% yield) as an oil and a mixture of 4 and its20-epimer 2 (126 mg, 43% yield) in ca. 1:3 ratio (by ¹H NMR). 4:[α]_(D)+40.8° (c 1.09, CHCl₃), ¹H NMR (500 MHz, CDCl₃) δ4.00 (1H, d,J=1.9 Hz, 8α-H), 3.70 (1H, dd, J=10.6, 3.2 Hz, 22-H), 3.43 (1H, dd,J=10.6, 7.0 Hz, 22-H), 0.94 (3H, d, J=6.7 Hz, 21-H₃), 0.927 (3H, s,18-H₃), 0.884 (9H, s, Si-t-Bu), 0.007 and −0.006 (each 3H, each s,SiMe₂); ¹³C NMR (125 MHz) δ69.30 (d, C-8), 66.83 (t, C-22), 53.02 (d),52.96 (d), 41.91 (s, C-13), 40.12 (t), 37.48 (d), 34.38 (t), 26.71(t),25.79 (q, SiCMe₃), 22.85 (t), 18.01 (s, SiCMe₃), 17.64 (t), 16.58 (q,C-21), 14.07 (q, C-18), −4.81 and −5.18 (each q, each Sie).

Preparation of(20R)-de-A,B-8β-(tert-butyldimethylsilyI)oxy-20-(iodomethyl)pregnane(5).

A solution of iodine (471 mg, 1.84mmol) in methylene chloride (30 mL)was slowly added to a solution of triphenylphosphine (482 mg, 1.84mmol)and imidazole (250 mg, 3.68mmol) in methylene chloride (15 mL) at 0° C.After 15 min. a solution of alcohol 4 (149 mg, 0.46 mmol) in methylenechloride (3 mL) was added into the mixture. After being stiring for 20min. at 0° C., followed by 18 h at room temperature, the reactionmixture was washed with water, dried (Na₂SO₄) and concentrated underreduced pressure. The residue was chrornatographed on silica gel withhexane/ethyl acetate (97:3) to give the desired iodide 5 (201 mg, 100%):[α]_(D)−0.30 (c 0.97, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ3.99 (1H, s,8α-H), 3.46 (1H, dd, J=9.5,2.9 Hz, 22-H), 3.18 (1H, dd, J =9.5, 6.4 Hz),1.88-1.74 (3H, m), 1.67 (1H, dm, J=13.9 Hz), 0.95 (3H, d, J=6.4 Hz,21-H₃), 0.918 (3H, s, 18-H₃), 0.882 (9H, s, Si-t-Bu), 0.008 and −0.008(each, 3H, each s, SiMe₂); ¹³C NMR (125 MHz) δ69.27 (d, C-8), 55.19 (d),52.69 (d), 41.99 (s, C-13), 40.48 (t), 36.15 (d), 34.24 (t), 26.90 (t),25.80 (q, SiCMe₃), 22.81 (t), 21.38 (q, C-21), 19.58 (t), 18.02 (s,SiCMe₃), 17.63 (t), 14.12 (q, C-18), −4.79 and −5.17 (each q, eachSiMe); MS (EI) m/z 436 (15, M⁺), 421 (8, M⁺-CH₃), 393 (9, M⁺-C₃H₇), 379(98, M⁺-t-Bu), 303 (65, M⁺-t-BuMe₂SiOH-H), 177 (70), 135 (70), 95 (55),75 (100); exact mass calculated for C₁₉H₃₇OSiI (M⁺) 436.1658, found436.1672.

Preparation of(20S)-de-A,B-8β-(tert-butyldimethyhsilyl)oxy-20-(3-isopropoxycarbonyl)propyl-pregnane(6).

A mixture of zinc powder (124 mg, 1.9 mmol), anhydrous pyridine (4 mL)and isopropyl acrylate (235 μL, 217 mg, 1.9 mmol) was warmed to 50° C.,then nickel(II) chloride hexahydrate (109 mg, 0.46 mmol) was added. Theresulting mixture was warmed to 65° C. and stirred for 2 h until itsgreen color turned to reddish brown one. After cooling to 0° C., asolution of iodide 5 (222 mg, 0.51 mmol) in anhydrous pyridine (3 mL)was added and the reaction mixture was stirred for 4 h at roomtemperature. The mixture was diluted with ethyl acetate (20 mL) and theresulting precipitate was filtered off through a pad of Celite. Thefiltrate was washed with 5% aq. HCl and brine, dried (Na₂SO₄) andconcentrated under reduced pressure. The residue was chromatographed onsilica gel with hexane and hexane/ethyl acetate (95:5) to give the ester6 (177 mg, 82%): [α]_(D)+19.7° (c 1.13, CHCl₃); ¹H NMR (400 MHz, CDCl₃)δ5.00 (1H, sep, J=6.3 Hz, OCHMe₂), 3.99 (1H, d, J=2.2 Hz, 8α-H), 2.23(1H, dd, J=7.4, 2.5 Hz, 24-H), 2.21 (1H, dd, J=6.8, 1.9 Hz, 24-H), 1.90(1H, dm, J=12.2 Hz), 1.22 (6H, d, J=6.3 Hz, OCHMe₂) 0.895 (3H, s,18-H₃), 0.881 (9H s, Si-t-Bu), 0.82 (3H, d, J=6.6 Hz, 21-H₃), 0.001 and−0.012 (each, 3H, each s, SiMe₂); ¹³C NMR (100 MHz) δ173.48 (s,COO-iPr), 69.45 (d, C-8), 67.31 (d, COOCHMe₂), 56.29 (d), 53.08 (d),42.16 (s, C-13), 40.64 (t), 35.05 (t), 34.71 (t), 34.51 (d), 34.44 (t),27.16 (t), 25.80 (q, SiCMe₃, 22.93 (t), 21.92 (t), 21.86 (q, COOCHMe₂),18.48 (q, C-21), 18.02 (t), 17.69 (s, SiCMe₃), 14.01 (q, C-18), −4.79and −5.16 (each q, each SiMe; MS (EI) m/z 424 (5, M⁺), 409 (15, M⁺-CH₃),381 (35, M⁺-C₃H₇), 367 (89, M³⁰ -t-Bu), 321 (39, M⁺-CH₃COOCHMe₂ -H), 307(85, M⁺-CH₃CH₂COOCHMe₂ -H), 283 (65), 265 (41), 249 (45), 233 (60), 215(73), 189 (70), 163 (78), 135 (86), 109 (70), 95 (79), 75 (100); exactmass calculated for C₂₅H₄₈O₃Si (M⁺) 424.3372, found 424.3371.

Preparation of(20S)-de-A,B-8β-(tert-butyldinethylsilyi)oxy-20-(4-hydroxybutyl)pregnane (7)

Lithium aluminium hydride (20 mg, 0.53 mmol) was added to a solution ofester 6 (118 mg, 0.28 mmol) in anhydrous TBF (5 mL) at 0° C. Thereaction mixture was stirred for 30 min at 0° C., then a cooling bathwas removed and the stirring was continued for additional 19 h at roomtemperature. The excess hydride was quenched by careful, successiveaddition of sat. aq. NH₄Cl. Methylene chloride (15 mL) and Celite (0.5g) were added and the slurry was stirred for 20 min. The aluminium saltswere separated by vacuum filtering the slurry through a Celite pad. Thesalts were repeatedly washed with methylene chloride. The filtrate wasdried (Na₂SO₄) and concentrated under reduced pressure. A residue waschromatographed on silica gel with hexane/ethyl acetate (90:10) toafford the alcohol 7 (96 mg, 93% yield) as a colorless oil:[α]_(D)+25.5° (c 1.0, CHCl₃); ¹H NMR (400 Mz, CDCl₃) δ3.99 (1H, d, J=2.1Hz, 8α-H), 3.64 (2H, t,-J=6.6 Hz, CH₂OH), 1.92 (1H, dm, J=12.3 Hz),0.907 (3H, s, 18-H₃), 0.886 (9H, S, Si-t-Bu), 0.81 (3H, d, J=6.6 Hz,21-H₃), 0.007 and −0.006 (each, 3H, each s, SiMe₂); ¹³CNMR (100 MHz)δ69.43 (d, C-8), 63.18 (t, C-25), 56.31 (d), 53.10 (d), 42.17 (s, C-13),40.65 (t), 35.05 (t), 34.70 (d), 34.45 (t), 33.20 (t), 27.17 (t), 25.79(q, SiCMe₂), 22.94 (t), 22.35 (t), 18.53 (q, C-21), 18.02 (s, SiCMe₃),17.71 (t), 14.03 (q, C-18), −4.81 and −5.17 (each q, each SiMe; MS (EI)m/z no M⁺, 325 (3, M⁺-C₃H₇), 311 (9, M⁺-C₄H₉), 269 (6, M⁺-C₆H₁₁O) 251(16, M⁺-H-t-BuSiMe₂H), 235 (25, M⁺-H-t-BuSiMe₂OH), 219 (29), 163 (46),135 (78), 109 (62), 75 (100); exact mass calculated for C₁₈H₃₅O₂Si(M⁺-C₄H₉) 311.2406, found 311.2397.

Preparation of(20S)-de-A,B8β-(tert-butyldimethylsilyl)oxy-20-butyl-pregnane (8)

To a stirred solution of the alcohol 7 (95 mg, 0.26 imnol),4-dimethylaminopyridine (5 mg, 0.04 mmol) and triethylamine (145 μL, 105mg, 1.04 mmol) in anhydrous methylene chloride (5 mL) p-toluenesulfonylchloride (68 mg, 0.36 mmol) was added at 0° C. A cooling bath wasremoved and stirring was continued for 22 h. Methylene chloride (20 mL)was added and the mixture was washed with a saturated aq.NaHCO₃solution, dried (Na₂SO₄) and concentrated under reduced pressure.A residue was dissolved in anhydrous THF (5 mL) and lithium aluminiumhydride (32 mg, 0.84 mmol) was added to the solution at 0° C. A coolingbath was removed and the mixture was stirred for 18 h at roomtemperature. The excess hydride was quenched by carefuil, successiveaddition of sat. aq. NH₄Cl. Methylene chloride (15 mL) and Celite (0.5g) were added and the slurry was stirred for 20 min. The aluminium saltswere separated by vacuum filtering the slurry through a Celite pad. Thesalts were repeatedly washed with methylene chloride. The filtrate wasdried (Na₂SO₄) and concentrated under reduced pressure. A residue waschromatographed on silica gel with hexane/ethyl acetate (97:3) to givethe product 8 (85 mg, 93% yield): [α]_(D)+25.30° (c 1.26, CHCl₃); ¹H NMR(400 MHz, CDCl₃) δ4.00 (1H, d, J=2.1 Hz, 8α-H), 1.95 (1H, dm, J=12.4Hz), 0.914 (3H, s, 18-H₃), 0.893 (9H, s, Si-t-Bu), 0.81 (3H, d, J=6.6Hz, 21-H₃), 0.013 and 0.000 (each 3H, each s, each SiMe); ¹³C NMR (100MHz) δ69.52 (d, C-8), 56.47 (d), 53.15 (d), 42.19 (s, C-13), 40.68 (t),35.02 (t), 34.79 (d), 34.52 (t), 28.56 (t), 27.21 (t), 25.81 (q, SiCe₃),23.09 (t), 22.99 (t), 18.62 (q, C-21), 18.05 (s, SiCMe₃), 17.75 (t),14.26 (q, C- 25), 14.02 (q, C-18), −4.79 and −5.16 (each q, each SiMe);MS (EI) m/z 352 (2, M⁺), 337 (4, M⁺-CH₃), 295 (81, M⁺-t-Bu), 253 (13,M⁺-C₆H₁₁,O), 219 (71, M⁺-H-t-BuSiMe₂OH), 177 (10), 135 (22), 75 (100);exact mass calculated for Cl₈H₃₅OSi (M⁺-C₄H₉) 295.2457, found 295.2454.

Preparation of (20S)-de-A,B 20-butyl-pregnan-8β-ol (9)

The protected alcohol 8 (84 mg, 0.24 mmol) was dissolved in anhydrousTHF (5 mL) and anhydrous methanol (5 mL). Hydrogen fluoride-pyridinecomplex (4 mL) was added followed at room temperature and the mixturewas stirred for 19 h. Ethyl acetate (20 mL) was added and the organicphase was washed with brine and water, dried (Na₂SO₄) and concentratedunder reduced pressure. The residue was diluted with hexane andchromatographed on silica gel with hexane to give the product 9 (17 mg,30% yield) as a colorless oil: ¹H NMR (500 MHz, CDCl₃) δ4.07 (1H, d,J=2.5 Hz, 8α-H), 1.98 (1H, dm J=13.1 Hz), 1.88-1.76 (3H, m), 0.927 (3H,s, 18-H₃), 0.89 (3H, t, J=7.1 Hz, 25-H₃), 0.81 (3H, d, J=6.6 Hz, 21-H₃);¹³C NMR (125 MHz) δ69.46 (d, C-8), 56.32 (d), 52.67 (d), 41.90 (s,C-13), 40.32 (t), 34.97 (t), 34.76 (d), 33.59 (t), 28.52 (t), 27.05 (t),23.08 (t), 22.42 (t), 18.56 (q, C-21), 17.49 (t), 14.23 (q, C-25), 13.77(q, C-18).

Preparation of (20S)-de-A,B-20-butyl-pregnan-8-one (10)

Pyridinium dichromate (118 mg, 314 μmol) was added to a solution of thealcohol. 9 (15 mg, 63 μmol) and pyridinium p-toluenesulfonate (2 mg, 8μmol) in anhydrous methylene chloride (5 mL). The resulting suspensionwas stirred at roorn temperature for 2 h. The reaction mixture wasfiltered through a Waters silica Sep-Pak cartridge (5 g) that was etherwashed with hexane/ethyl acetate (95:5). After removal of solvents theketone 10 (12 mg, 81% yield) was obtained as a colorless oil: ¹H NMR(400 MHz, CDCl₃) δ2.45 (1H, dd, J=11.5, 7.6 Hz), 2.32-2.16 (2H, m), 0.90(3H, t, J=6.9 Hz, 25-H₃), 0.85 (3H, d, J=6.1 Hz, 21-H₃), 0.634 (3H, s,18-H₃); ¹³C NMR (100 MHz) δ212.14 (C-8), 62.01 (C-14), 56.24, 49.96(C-13), 40.96, 38.86, 35.18, 34.87, 28.43, 27.15, 24.06, 23.03, 18.94(C-21), 18.51, 14.19 (C-25), 12.72 (C-18).

Preparation of (20S)-2-methylene-19,26,27-trinor-α-hydroxyvitamin D₃(13).

To a solution of phosphine oxide 11 (60 mg, 103 μmol) in anhydrous THF(600 μL) at −20° C.0 was slowly added PhLi (1.8 M in cyclohexane-ether,60 μL, 108 μmol) under argon with stirring. The solution turned deeporange. After 30 min the rnixture was cooled to −78° C. and a precooled(−78° C.) solution of ketone 10 (12 mg, 51 μmol) in anhydrous THF (200μL) was slowly added. The mixture was stirred under argon at -78 ° C.for 3 h and at 0° C. for 18 h. Ethyl acetate was added, and the organicphase was. washed with brine, dried (Na₂SO₄) and evaporated. The residuewas dissolved in hexane and applied on a Waters silica Sep-Pak cartridge(2 g). The cartridge was washed with hexane and hexane/ethyl acetate(99.5:0.5) to give 19-norvitamin derivative 12 (13 mg). The Sep-Pak wasthen washed with hexane/ethyl acetate (96:4) to recover the unchangedC,D-ring ketone 10 (6 mg, 25 μmol), and with ethyl acetate to recoverdiphenylphosphine oxide 11 (56 mg). The protected vitamin 12 was furtherpurified by HPLC (9.4×250 mm Zorbax-Silica column, 4 mL/min) usinghexane/2-propanol (99.9:0.1) solvent system. Pure compound 12 (8.3 mg,53% yield) was eluted at R_(t)=3.2 min as a colorless oil: MS (EI) m/z600 (14, M⁺), 585 (4, M⁺-Me), 543 (11, M⁺-C₄H₉), 468 (100,M⁺-t-BUMe₂SiOH), 366 (43), 323 (9), 257 (13), 234 (16), 147 (24), 73(97); exact mass calculated for C₃₇H₆O₂Si₂ (M⁺) 600.4758, found600.4742.

Protected vitamin 12 (8 mg, 13 μmol) was dissolved in anhydrous THF (4mL) and a solution of tetrabutylaionium fluoride (1 M in THF, 100 μL,100 μmol) was added, followed by freshly activated molecular sieves 4A(300 mg). The mixture was stirred under argon at room temperature for 4h, then diluted with 2 mL of hexane/ethyl acetate (9:1) and applied on aWaters silica Sep-Pak cartridge (2 g). Elution with the same solventsystem gave the crude product 13 that was further purified by HPLC(9.4×250 mm Zorbax-Silica column, 4 mL/min) using hexane/2-propanol(9:1) solvent system. Analytically pure 2-methylene-19-norvitamin 13(3.59 mg, 74% yield) was collected at R_(t)=6.4 min. as a colorless oil:UV (in EtOH) δ_(max), 261, 251, 243 nm; ¹H NMR (750 MHz, CDCl₃) δ6.36and 5.89 (1H and 1H, each d, J=11.2 Hz, 6-and 7-H), 5.11 and 5.09 (each1H, each s, =CH₂), 4.47 (2H, m, 1β-and 3α-H), 2.84 (1H, dd, J=13.3, 4.4Hz, 10β-H), 2.82 (1H, br d, J=12.3 Hz, 9β-H), 2.58 (1H, dd, J=13.3, 3.4Hz, 4α-H), 2.32 (1H, dd, J=13.3, 6.1 Hz, 4β-H), 2.30 (1H, dd, J=13.3,8.4 Hz, 10α-H), 2.05-1.95 (2H, m), 1.90-1.82 (1H, m), 0.89 (3H, t, J=7.1Hz, 25-H₃), 0.84 (3H, d, J=6.5 Hz, 21-H₃), 0.552 (3H, s, 18-H₃); ¹³C NMR(100 MHz) δ151.97 (s, C-2), 143.55 (s, C-8), 130.29 (s, C-5), 124.30 (d,C-6), 115.24 (d, C-7), 107.71 (t, =CH₂), 71.82 and 70.70 (each d, C-1and C-3), 56.36 (d), 56.22 (d), 45.84 (s, C-13), 45.79 (t), 40.34 (t),38.16 (t), 35.45 (d), 35.29 (t), 28.98 (t), 28.55 (t), 27.29 (t), 23.51(t), 23.08 (t), 22.17 (t), 18.59 (q, C-21), 14.23 (q, C-25), 12.33 (q,C-18); MS (EI) m/z 372 (100, M⁺), 354 (4, M⁺-H₂O), 324 (15,M⁺-H₂O-C₂H₆), 287 (60, M⁺C6H₁₃), 269 (22, M⁺-C₆H₁₃-H₂O), 251 (18,M⁺-C₆H₁₃-2H₂0), 231 (22), 219 (35),;147 (46), 135(76), 119 (27), 107(61); exact mass calculated for C₂₅H₄₀O₂ (M⁺) 372.3028 found 372.3039.

Preparation of (20S)-2α-methyl-19,26,27-trinor-1α-hydroxyvitamiin D₃(14) and (20S)-2β-methyl-19,26,27-trinor-lo-hydroxyvitaamin D₃ (15).

Tris(triphenylphosphine)rhodium (I) chloride (3.7 mg, 4 pnol) was addedto dry benzene (2.5 mL) presaturated with hydrogen. The mixture wasstirred at room temperature until a homogeneous solution was formed (ca.45 mnin). A solution of vitamin 13 (1.4 mg, 3.8 ,μmol) in dry benzene(400+100 μL) was then added and the reaction was allowed to proceedunder a continuous stream of hydrogen for 3 h. Benzene was removed undervacuum, the residue was redissolved in hexane/ethyl acetate (1:1) andapplied on a Waters silica Sep-Pak cartridge (2 g). A mixture of2-methyl vitamins was eluted with the same solvent system. The compoundswere further purified by HPLC (9.4×250 mm Zorbax-Silica column, 4mL/min) using hexane/2-propanol (9:1) solvent system. The mixture of2-methyl-19-norvitamins 14 and 15 gave a single peak at R_(t)=7.0 min.Separation of both epimers was achieved by reversed-phase HPLC (9.4×250mm Zorbax Eclipse XDB-C18 column, 3 mL/min) using methanol/water (9:1)solvent system. 2β-Methyl vitamin 15 (266 μg, 19% yield) was collectedat R_(t)=15.9 min. and its 2α-epimer 14 (398 μg, 28% yield) atR_(t)=18.2 min.

2α-Methyl analog 14: UV (in EtOH)δ_(max) 260, 250, 242 nm; ¹H NMR (500MHz , CDCl₃) δ6.37 and 5.82 (1H and 1H, each d, J=11.3 Hz, 6- and 7-H),3.96 (1H, m, w/2=14 Hz, 1β-H), 3.61 (1H, m, w/2'21 Hz, 3α-H), 2.80 (2H,br m, 9β- and 10α-H), 2.60 (1H, dd, J=13.2, 4.4 Hz, 4α-H), 2.22 (1H, brd, J=12.7 Hz, 10,β-H), 2.13 (1H, ˜t, J˜12.0 Hz, 4,β-H), 1.133 (3H, d,J=6.8 Hz, 2α-CH₃), 0.887 (3H, t, J=7.1 Hz, 25-H₃), 0.829 (3H, d, J=6.5Hz, 21-H₃), 0.529 (3H, s, 18-H₃); MS (El) m/z 374 (100, M⁺), 317 (15,M⁺-C₄H₉), 289 (40, M⁺-C₆H₁₃), 271(17, M⁺-C₆H₁₃-H₂O), 253 (17, M⁺-C₆H₁₃-2H₂O ), 231 (29), 219 (47), 147 (31), 129 (42); exact masscalculated for C₂₅H₄₂O₂ (M⁺) 374.3185 found 374.3186.

2,-Methyl analog 15: UV (in EtOH) δ_(max) 260, 250, 242 nm; ¹H NMR (500MHz, CDCl₃) 8 6.26 and 5.87 (1H and 1H, each d, J=11.1 Hz, 6-H and 7-H),3.90 (1H, m, w/2 =15 Hz, 3α-H), 3.50 (1H, m, w/2=26 Hz, 1β-H), 3.08 (1H,dd, J=12.4, 4.6 Hz, 10β-H), 2.80 (1H, dd, J=12.4, 4.1 Hz, 9β-H), 2.43(1H, br d, J=ca 14 Hz, 4α-H), 2.34 (1H, dd, J=14.0, 2.8 Hz, 4β-H), 1.142(3H, d, J=6.8 Hz, 2β-CH₃), 0.997 (3H, t, J=7.1 Hz, 25-H₃), 0.833 (3H, d,J=6.5 Hz, 21-H₃), 0.541 (3H, s, 18-H₃); MS (EI) m/z 374 (75, M⁺), 317(12, M⁺- C₄H₉), 289 (28, M⁺- C₆H₁₃), 271 (13, M⁺-C₆H₁₃-H₂O), 253 (12,M⁺-C₆H₁₃-2H₂O), 219 (32), 149 (45), 135 (38), 81 (52), 69 (100); exactmass calculated for C₂₅H₄₂O₂ (M⁺) 374.3185, found 374.3172.

BIOLOGICAL ACTIVITY OF 2α-METHYL AND25-METHYL ANALOGS OF19,26,27-TRINOR-(20S)-1α-HYDROXYVITAMIN D₃

With regard to the 2β-methyl analog C-19, the introduction of a methylgroup in an alpha orientation to the 2-position and the removal of thetwo methyl groups at the 26 and 27 positions in the side chain of1α-hydroxy-19-nor-(20S)-vitamin D₃ had little effect on binding to thefull length recombinant rat vitamin D receptor, as compared to1α,25-dihydroxyvitamin D₃. The compound C-19 bound similar to thereceptor as compared to the standard 1,25-(OH)₂D₃ (FIG. 1). It might beexpected from these results that compound C-19 would have equivalentbiological activity. Surprisingly, however, compound C-19 is a highlyselective analog with unique biological activity.

FIG. 4 demonstrates that C-19 has very little bone calcium mobilizationactivity, as compared to 1,25(OH)₂D₃. Thus, C-19 may be characterized ashaving little, if any, calcemic activity.

FIG. 2 illustrates that C-19 is almost as potent as 1,25(OH)₂D₃ on HL-60differentiation, making it an excellent candidate for the treatment ofpsoriasis and cancer, especially against leukemia, colon cancer, breastcancer, skin cancer and prostate cancer. In addition, due to itsrelatively high cell differentiation activity, this compound provides atherapeutic agent for the treatment of various skin conditions includingwrinkles, lack of adequate dermal hydration, i.e. dry skin, lack ofadequate skin firmness, i.e. slack skin, and insufficient sebumsecretion. Use of this compound thus not only results in moisturizing ofskin but also improves the barrier function of skin.

FIG. 3 illustrates that the compound C-19 has transcriptional activitybut significantly less than 1α,25-dihydroxyvitamin D₃ in bone cells.This result, together with the cell differentiation activity of FIG. 2,suggests that C-19 will be very effective in psoriasis because it hasdirect cellular activity in causing cell differentiation and insuppressing cell growth. These data also indicate that C-19 may havesignificant activity as an anti-cancer agent, especially againstleukemia, colon cancer, breast cancer, skin cancer and prostate cancer.

The strong activity of C-19 on HL-60 differentiation suggest it will beactive in suppressing growth of parathyroid glands and in thesuppression of the preproparathyroid gene.

With regard to the 2β-methyl analog N-16, the introduction of a methylgroup in a beta orientation to the 2-position and the removal of the twomethyl groups at the 26 and 27 positions in the side chain of1α-hydroxy-19-nor-(20S)-vitamin D₃ resulted in this compound havingrelatively low binding to the full length recombinant rat vitamin Dreceptor, as compared to 1α,25-dihydroxyvitamin D₃. The compound N-16 isthus relatively inactive in binding to the receptor as compared to thestandard 1,25-(OH)₂D₃ (FIG. 1). The data in FIG. 1 thus indicates thatcompound N-16 would be considered relatively inactive in this regard.Surprisingly, however, compound N-16 retains some unique biologicalactivities.

FIG. 2 illustrates that N-16 has significant HL-60 cell differentiationactivity, as compared to 1,25(OH)₂D₃, making it an excellent candidatefor the treatment of psoriasis and cancer, especially against leukemia,colon cancer, breast cancer, skin cancer and prostate cancer. Inaddition, due to its cell differentiation activity, this compoundprovides a therapeutic agent for the treatment of various skinconditions including wrinkles, lack of adequate dermal hydration, i.e.dry skin, lack of adequate skin firmness, i.e. slack skin, andinsufficient sebum secretion. Use of this compound thus not only resultsin moisturizing of skin but also improves the barrier function of skin.

FIG. 3 illustrates that the compound N-16 also has significanttranscriptional activity as compared to 1α,25-dihydroxyvitamin D₃ inbone cells. This result, together with the cell differentiation activityof FIG. 2, suggests that N-16 will be very effective in psoriasisbecause it has direct cellular activity in causing cell differentiation,altering gene transcription and in suppressing cell growth. These dataalso indicate that N-16 may have significant activity as an anti-canceragent, especially against leukemia, colon cancer, breast cancer, skincancer and prostate cancer.

EXPERIMENTAL METHODS

Vitamin D Receptor Binding

Test Material

Protein Source

Full-length recombinant rat receptor was expressed in E. coli BL21 (DE3)Codon Plus RIL cells and purified to homogeneity using two differentcolumn chromatography systems. The first system was a nickel affinityresin that utilizes the C-terminal histidine tag on this protein. Theprotein that was eluted from this resin was further purified using ionexchange chromatography (S-Sepharose Fast Flow). Aliquots of thepurified protein were quick frozen in liquid nitrogen and stored at −80°C. until use. For use in binding assays, the protein was diluted inTEDK₅₀ (50 mM Tris, 1.5 mM EDTA, pH7.4, 5 mM DTT, 150 mM KCl) with 0.1%Chaps detergent. The receptor protein and ligand concentration wereoptimized such that no more than 20% of the added radiolabeled ligandwas bound to the receptor.

Study Drugs

Unlabeled ligands were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry (1,25(OH)₂D₃: molar extinctioncoefficient =18,200 and λ_(max)=265 nm; Analogs: molar extinctioncoefficient =42,000 and λ_(max)=252 nm). Radiolabeled ligand(³H-1,25(OH)₂D₃, ˜159 Ci/mmole) was added in ethanol at a finalconcentration of 1 nM.

Assay Conditions

Radiolabeled and unlabeled ligands were added to 100 mcl of the dilutedprotein at a final ethanol concentration of ≦10%, mixed and incubatedovernight on ice to reach binding equilibrium. The following day, 100mcl of hydroxylapatite slurry (50%) was added to each tube and mixed at10-minute intervals for 30 minutes. The hydroxylapaptite was collectedby centrifugation and then washed three times with Tris-EDTA buffer (50mM Tris, 1.5 mM EDTA, pH 7.4) containing 0.5% Titron X-100. After thefmal wash, the pellets were transferred to scintillation vialscontaining 4 ml of Biosafe II scintillation cocktail, mixed and placedin a scintillation counter. Total binding was determined from the tubescontaining only radiolabeled ligand.

HL-60 Differentiation

Test Material

Study Drugs

The study drugs were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry. Serial dilutions were prepared sothat a range of drug concentrations could be tested without changing thefinal concentration of ethanol (<0.2%) present in the cell cultures.

Cells

Human promyelocytic leukemia (HL60) cells were grown in RPMI-1640 mediumcontaining 10% fetal bovine serum. The cells were incubated at 37° C. inthe presence of 5% CO₂.

Assay Conditions

HL60 cells were plated at 1.2×10⁵ cells/ml. Eighteen hours afterplating, cells in duplicate were treated with drug. Four days later, thecells were harvested and a nitro blue tetrazolium reduction assay wasperformed (Collins et al., 1979; J. Exp. Med. 149:969-974). Thepercentage of differentiated cells was determined by counting a total of200 cells and recording the number that contained intracellularblack-blue formazan deposits. Verification of differentiation tomonocytic cells was determined by measuring phagocytic activity (datanot shown).

In vitro Transcription Assay

Transcription activity was measured in ROS 17/2.8 (bone) cells that werestably trans fected with a 24-hydroxylase (24Ohase) gene promoterupstream of a luciferase reporter gene (Arbour et al., 1998). Cells weregiven a range of doses. Sixteen hours after dosing the cells wereharvested and luciferase activities were measured using a luminometer.

RLU=Relative Luciferase Units.

Intestinal Calcium Transport and Bone Calcium Mobilization

Male, weanling Sprague-Dawley rats were placed on Diet 11 (0.47% Ca)diet +AEK for one week followed by Diet 11 (0.02% Ca) +AEK for 3 weeks.The rats were then switched to a diet containing 0.47% Ca for one weekfollowed by two weeks on a diet containing 0.02% Ca. Dose administrationbegan during the last week on 0.02% calcium diet. Four consecutive ipdoses were given approximately 24 hours apart. Twenty-four hours afterthe last dose, blood was collected from the severed neck and theconcentration of serum calcium determined as a measure of bone calciummobilization. The first 10 cm of the intestine was also collected forintestinal calcium transport analysis using the everted gut sac method.

INTERPRETATION OF DATA

VDR Binding, HL60 Cell differentiation, and Transcription Activity.

With regard to the 2α-methyl analog, C-19 (K_(i)=2.2×10⁻¹⁰M) is similarto the natural hormone 1α,25-dihydroxyvitamin D₃ (K_(i)=4.4×10⁻¹¹ M) inits ability to compete with [³H]−1,25(OH)₂D₃ for binding to thefull-length recombinant rat vitamin D receptor (FIG. 1). There is alsolittle difference between C-19 (EC₅₀=8.2×10⁻⁹M) in its ability (efficacyor potency) to promote HL60 differentiation as compared to1α,25-dihydroxyvitamin D₃ (EC₅₀=2.9×10⁻⁹M) (See FIG. 2). Also, compoundC-19 (EC₅₀=1×10⁻⁸M) has significant transcriptional activity in bonecells, but significantly less than 1α,25-dihydroxyvitamin D₃(EC₅₀=1.9×10⁻¹⁰M) (See FIG. 3).

With regard to the 2β-methyl analog, N-16 (K_(i)=1×10⁻⁸M) is relativelyinactive in vitamin D receptor binding, i.e. has little ability tocompete with [³H]-1,25(OH)₂D₃ for binding to the full length recombinantrat vitamin D receptor (FIG. 1). However, N=16 (EC₅₀=1×10⁻⁷M) canpromote HL-60 differentiation (See FIG. 2). Also, compound N-16(EC₅₀=1×10⁻⁷M) has transcriptional activity in bone cells (See FIG. 3).

These results suggest that C-19 and N-16 will both be very effective inpsoriasis because they both have direct cellular activity in causingcell differentiation and in suppressing cell growth. These data alsoindicate that C-19 and N-16 will both have significant activity asanti-cancer agents, especially against leukemia, colon cancer, breastcancer, skin cancer and prostate cancer, as well as against skinconditions such as dry skin (lack of dermal hydration), undue skinslackness (insufficient skin firmness), insufficient sebum secretion andwrinkles.

Both C-19 and N-16 would also be expected to be very active insuppressing secondary hyperparathyroidism.

Calcium Mobilization from Bone in Vitamin D-deficient Animals.

Using vitamin D-deficient rats on a low calcium diet (0.02%), theactivities of C-19 and 1,25(OH)₂D₃ in intestine and bone were tested. Asexpected, the native hormone (1,25(OH)₂D₃) increased serum calciumlevels at all dosages (FIG. 4). FIG. 4, however, also shows that C-19has little, if any, activity in mobilizing calcium from bone.Administration of C-19 at 780 pmol/day for 4 consecutive days did notresult in mobilization of bone calcium, and increasing the amount ofC-19 to 2340 pmol/day and then to 7020 pmol/day was also without anysubstantial effect.

These results illustrate that C-19 and N-16 are both excellentcandidates for numerous human therapies as described herein, and thatthey both may be particularly useful in a number of circumstances suchas suppression of secondary hyperparathyroidism of renal osteodystrophy,autoimmune diseases, cancer, and psoriasis. C-19 and N-16 are bothexcellent candidates for treating psoriasis because: (1) they havesignificant transcription activity and cellular differentiationactivity; (2) they are devoid of hypercalcemic liability unlike1,25(OH)₂D₃; and (3) they are easily synthesized. Also, since C-19 hassignificant binding activity to the vitamin D receptor, but has littleability to raise blood serum calcium, it may also be particularly usefulfor the treatment of secondary hyperparathyroidism of renalosteodystrophy.

These data also indicate that the compounds C-19 and N-16 of theinvention may both be especially suited for treatment and prophylaxis ofhuman disorders which are characterized by an imbalance in the immunesystem, e.g. in autoimmune diseases, including multiple sclerosis,lupus, diabetes mellitus, host versus graft rejection, and rejection oforgan transplants; and additionally for the treatment of inflammatorydiseases, such as rheumatoid arthritis, asthma, and inflammatory boweldiseases such as celiac disease, ulcerative colitis and Crohn's disease.Acne, alopecia and hypertension are other conditions which may betreated with the compounds C-19 and N-16 of the invention.

The compounds of the invention of formula I, and particularly formulaIa, are also useful in preventing or treating obesity, inhibitingadipocyte differentiation, inhibiting SCD-1 gene transcription, and/orreducing body fat in animal subjects. Therefore, in some embodiments, amethod of preventing or treating obesity, inhibiting adipocytedifferentiation, inhibiting SCD-1 gene transcription, and/or reducingbody fat in an animal subject includes administering to the animalsubject, an effective amount of one or more of the compounds or apharmaceutical composition that includes one or more of the compounds offormula I. Administration of the compound or the pharmaceuticalcompositions to the subject inhibits adipocyte differentiation, inhibitsgene transcription, and/or reduces body fat in the animal subject. Theanimal may be a human, a domestic animal such as a dog or a cat, or anagricultural animal, especially those that provide meat for humanconsumption, such as fowl like chickens, turkeys, pheasant or quail, aswell as bovine, ovine, caprine, or porcine animals.

For prevention and/or treatment purposes, the compounds of thisinvention defined by formula I may be formulated for pharmaceuticalapplications as a solution in innocuous solvents, or as an emulsion,suspension or dispersion in suitable solvents or carriers, or as pills,tablets or capsules, together with solid carriers, according toconventional methods known in the art. Any such formulations may alsocontain other pharmaceutically-acceptable and non-toxic excipients suchas stabilizers, anti-oxidants, binders, coloring agents or emulsifyingor taste-modifying agents.

The compounds of formula I, and particularly C-19 and/or N-16, may beadministered orally, topically, parenterally, rectally, nasally,sublingually or transdermally. The compounds may be advantageouslyadministered by injection or by intravenous infusion or suitable sterilesolutions, or in the form of liquid or solid doses via the alimentarycanal, or in the form of creams, ointments, patches, or similar vehiclessuitable for transdermal applications. A dose of from 0.01 μg to 1000 μgper day of compounds I, and particularly compounds C-19 and/or N-16,preferably from about 0.1 μg to about 500 μg per day, is appropriate forprevention and/or treatment purposes, such dose being adjusted accordingto the disease to be treated, its severity and the response of thesubject as is well understood in the art. Since the compounds exhibitspecificity of action, each may be suitably administered alone, ortogether with graded doses of another active vitamin D compound—e.g.1α-hydroxyvitamin D₂ or D₃, or 1α,25-dihydroxyvitamin D₃—in situationswhere different degrees of bone mineral mobilization and calciumtransport stimulation is found to be advantageous.

Compositions for use in the above-mentioned treatments comprise aneffective amount of compounds I, and particularly compounds C-19 and/orN-16 as defined by the above formula Ia and lb as the active ingredient,and a suitable carrier. An effective amount of such compounds for use inaccordance with this invention is from about 0.01 μg to about 1000 μgper gm of composition, preferably from about 0.1 μg to about 500 μg pergram of composition, and may be administered topically, transdermally,orally, rectally, nasally, sublingually or parenterally in dosages offrom about 0.01 μg/day to about 1000 μg/day, and preferably from about0.1 μg/day to about 500 μg/day.

The compounds I, and particularly compounds C-19 and/or N-16 may beformulated as creams, lotions, ointments, topical patches, pills,capsules or tablets, suppositories, aerosols, or in liquid form assolutions, emulsions, dispersions, or suspensions in pharmaceuticallyinnocuous and acceptable solvent or oils, and such preparations maycontain in addition other pharmaceutically innocuous or beneficialcomponents, such as stabilizers, antioxidants, emulsifiers, coloringagents, binders or taste-modifying agents.

The compounds I, and particularly compounds C-19 and/or N-16 may beadvantageously administered in amounts sufficient to effect thedifferentiation of promyelocytes to normal macrophages. Dosages asdescribed above are suitable, it being understood that the amounts givenare to be adjusted in accordance with the severity of the disease, andthe condition and response of the subject as is well understood in theart.

The formulations of the present invention comprise an active ingredientin association with a pharmaceutically acceptable carrier therefore andoptionally other therapeutic ingredients. The carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulations and not deleterious to the recipient thereof.

Formulations of the present invention suitable for oral administrationmay be in the form of discrete units as capsules, sachets, tablets orlozenges, each containing a predetermined amount of the activeingredient; in the form of a powder or granules; in the form of asolution or a suspension in an aqueous liquid or non-aqueous liquid; orin the form of an oil-in-water emulsion or a water-in-oil emulsion.

Formulations for rectal administration may be in the form of asuppository incorporating the active ingredient and carrier such ascocoa butter, or in the form of an enema.

Formulations suitable for parenteral administration convenientlycomprise a sterile oily or aqueous preparation of the active ingredientwhich is preferably isotonic with the blood of the recipient.

Formulations suitable for topical administration include liquid orsemi-liquid preparations such as liniments, lotions, applicants,oil-in-water or water-in-oil emulsions such as creams, ointments orpastes; or solutions or suspensions such as drops; or as sprays.

Four nasal administration, inhalation of powder, self-propelling orspray formulations, dispensed with a spray can, a nebulizer or anatomizer can be used. The formulations, when dispensed, preferably havea particle size in the range of 10 to 100μ.

The formulations may conveniently be presented in dosage unit form andmay be prepared by any of the methods well known in the art of pharmacy.By the term “dosage unit” is meant a unitary, i.e. a single dose whichis capable of being administered to a patient as a physically andchemically stable unit dose comprising either the active ingredient assuch or a mixture of it with solid or liquid pharmaceutical diluents orcarriers.

1. A compound having the formula:

where the methyl group attached to carbon 2 may have an R or Sconfiguration, and where X₁ and X₂, which may be the same or different,are each selected from hydrogen or a hydroxy-protecting group.
 2. Thecompound of claim 1 wherein X₂ is hydrogen.
 3. The compound of claim 1wherein X₁ and X₂ are both hydrogen.
 4. The compound of claim 1 whereinX₁ and X₂ are both t-butyldimethylsilyl.
 5. A pharmaceutical compositioncontaining an effective amount of at least one compound as claimed inclaim 1 together with a pharmaceutically acceptable excipient.
 6. Thepharmaceutical composition of claim 5 wherein said effective amountcomprises from about 0.01 μg to about 1000 μg per gram of composition.7. The pharmaceutical composition of claim 5 wherein said effectiveamount comprises from about 0.1 μg to about 500 μg per gram ofcomposition.
 8. 2α-methyl- 19,26,27-trinor-(20S)-1α-hydroxyvitamin D₃having the formula:


9. A pharmaceutical composition containing an effective amount of2α-methyl -19,26,27-trinor-(20S)-1α-hydroxyvitamin D₃ together with apharmaceutically acceptable excipient.
 10. The pharmaceuticalcomposition of claim 9 wherein said effective amount comprises fromabout 0.01 μg to about 1000 μg per gram of composition.
 11. Thepharmaceutical composition of claim 9 wherein said effective amountcomprises from about 0.1 μg to about 500 μg per gram of composition. 12.A method of treating psoriasis comprising administering to a subjectwith psoriasis an effective amount of a compound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 13. The method of claim 12wherein the compound is administered orally.
 14. The method of claim 12wherein the compound is administered parenterally.
 15. The method ofclaim 12 wherein the compound is administered transdermally.
 16. Themethod of claim 12 wherein the compound is administered topically. 17.The method of claim 12 wherein the compound is administered rectally.18. The method of claim 12 wherein the compound is administered nasally.19. The method of claim 12 wherein the compound is administeredsublingually.
 20. The method of claim 12 wherein the compound isadministered in a dosage of from about 0.01 μg/day to about 1000 μg/day.21. The method of claim 12 wherein the compound is 2α-methyl- 19,26,27-trinor-(20S)- 1 cc-hydroxyvitamin D₃ having the formula:


22. A method of treating a disease selected from the group consisting ofleukemia, colon cancer, breast cancer, skin cancer or prostate cancercomprising administering to a subject with said disease an effectiveamount of a compound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 23. The method of claim 22wherein the compound is administered orally.
 24. The method of claim 22wherein the compound is administered parenterally.
 25. The method ofclaim 22 wherein the compound is administered transdermally.
 26. Themethod of claim 22 wherein the compound is administered rectally. 27.The method of claim 22 wherein the compound is administered nasally. 28.The method of claim 22 wherein the compound is administeredsublingually.
 29. The method of claim 22 wherein the compound isadministered in a dosage of from about 0.01 μg/day to about 1000 μg/day.30. The method of claim 22 wherein the compound is2α-methyl-19,26,27-trinor -(20S)- 1α-hydroxyvitamin D₃ having theformula:


31. A method of treating an autoimmune disease selected from the groupconsisting of multiple sclerosis, lupus, diabetes mellitus, host versusgraft rejection, and rejection of organ transplants, comprisingadministering to a subject with said disease an effective amount of acompound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 32. The method of claim 31wherein the compound is administered orally.
 33. The method of claim 31wherein the compound is administered parenterally.
 34. The method ofclaim 31 wherein the compound is administered transdermally.
 35. Themethod of claim 31 wherein the compound is administered rectally. 36.The method of claim 31 wherein the compound is administered nasally. 37.The method of claim 31 wherein the compound is administeredsublingually.
 38. The method of claim 31 wherein the compound isadministered in a dosage of from about 0.01 μg/day to about 1000 μg/day.39. The method of claim 31 wherein the compound is2α-methyl-19,26,27-trinor -(20S)-1α-hydroxyvitamin D₃ having theformula:


40. A method of treating an inflammatory disease selected from the groupconsisting of rheumatoid arthritis, asthma, and inflammatory boweldiseases, comprising administering to a subject with said disease aneffective amount of a compound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 41. The method of claim 40wherein the compound is administered orally.
 42. The method of claim 40wherein the compound is administered parenterally.
 43. The method ofclaim 40 wherein the compound is administered transdermally.
 44. Themethod of claim 40 wherein the compound is administered rectally. 45.The method of claim 40 wherein the compound is administered nasally. 46.The method of claim 40 wherein the compound is administeredsublingually.
 47. The method of claim 40 wherein the compound isadministered in a dosage of from about 0.01 μg/day to about 1000 μg/day.48. The method of claim 40 wherein the compound is 2α-methyl-19,26,27-trinor -(20S)-1α-hydroxyvitamin D₃ having the formula:


49. A method of treating a skin condition selected from the groupconsisting of wrinkles, lack of adequate skin firmness, lack of adequatedermal hydration and insufficient sebum secretion which comprisesadministering to a subject with said skin condition an effective amountof a compound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 50. The method of claim 49wherein the compound is administered orally.
 51. The method of claim 49wherein the compound is administered parenterally.
 52. The method ofclaim 49 wherein the compound is administered transdermally.
 53. Themethod of claim 49 wherein the compound is administered topically. 54.The method of claim 49 wherein the compound is administered rectally.55. The method of claim 49 wherein the compound is administered nasally.56. The method of claim 49 wherein the compound is administeredsublingually.
 57. The method of claim 49 wherein the compound isadministered in a dosage of from about 0.01 μg/day to about 1000 μg/day.58. The method of claim 49 wherein the compound is 2α-methyl-19,26,27-trinor-(20S)-1α-hydroxyvitamin D₃ having the formula:


59. A method of treating renal osteodystrophy comprising administeringto a subject with renal osteodystrophy an effective amount of a compoundhaving the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 60. The method of claim 59wherein the compound is administered orally.
 61. The method of claim 59wherein the compound is administered parenterally.
 62. The method ofclaim 59 wherein the compound is administered transdermally.
 63. Themethod of claim 59 wherein the compound is administered rectally. 64.The method of claim 59 wherein the compound is administered nasally. 65.The method of claim 59 wherein the compound is administeredsublingually.
 66. The method of claim 59 wherein the compound isadministered in a dosage of from about 0.01 μg/day to about 1000 μg/day.67. The method of claim 59 wherein the compound is2α-methyl-19,26,27-trinor -(20S)-1α-hydroxyvitamin D₃ having theformula:


68. A method of treating or preventing obesity of an animal comprisingadministering to an animal in need thereof an effective amount of acompound having the formula:

wherein X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 69. The method of claim 68wherein the compound is administered orally.
 70. The method of claim 68wherein the compound is. administered parenterally.
 71. The method ofclaim 68 wherein the compound is administered transdermally.
 72. Themethod of claim 68 wherein the compound is administered rectally. 73.The method of claim 68 wherein the compound is administered nasally. 74.The method of claim 68 wherein the compound is administeredsublingually.
 75. The method of claim 68 wherein the compound isadministered in a dosage of from about 0.01μg/day to about 1000 μg/day.76. The method of claim 68 wherein the compound is2α-methyl-19,26,27-trinor -(20S)-1α-hydroxyvitamin D₃ having theformula:


77. The method of claim 68 wherein the animal is a human.
 78. The methodof claim 68 wherein the animal is a domestic animal.
 79. The method ofclaim 68 wherein the animal is an agricultural animal. 80.2β-methyl-19,26,27-trinor-(20S)-1β-hydroxyvitamin D₃ having the formula:


81. A pharmaceutical composition containing an effective amount of2β-methyl-19,26,27-trinor-(20S)-1α-hydroxyvitamin D₃ together with apharmaceutically acceptable excipient.
 82. The pharmaceuticalcomposition of claim 81 wherein said effective. amount comprises fromabout 0.01 μg to about 1000 μg per gram of composition.
 83. Thepharmaceutical composition of claim 53 wherein said effective amountcomprises from about 0.1 μg to about 500 μg per gram of composition. 84.A method of treating psoriasis comprising administering to a subjectwith psoriasis an effective amount of a compound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 85. The method of claim 84wherein the compound is administered orally.
 86. The method of claim 84wherein the compound is administered parenterally.
 87. The method ofclaim 84 wherein the compound is administered transdermally.
 88. Themethod of claim 84 wherein the compound is administered topically. 89.The method of claim 84 wherein the compound is administered rectally.90. The method of claim 84 wherein the compound is administered nasally.91. The method of claim 84 wherein the compound is administeredsublingually.
 92. The method of claim 84 wherein the compound isadministered in a dosage of from about 0.0 μg/day to about 1000 μg/day.93. The method of claim 84 wherein the compound is2β-methyl-19,26,27-trinor -(20S)-1α-hydroxyvitamin D₃ having theformula:


94. A method of treating a disease selected from the group consisting ofleukemia, colon cancer, breast cancer, skin cancer or prostate cancercomprising administering to a subject with said disease an effectiveamount of a compound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 95. The method of claim 94wherein the compound is administered orally.
 96. The method of claim 94wherein the compound is. administered parenterally.
 97. The method ofclaim 94 wherein the compound is administered transdermally.
 98. Themethod of claim 94 wherein the compound is administered rectally. 99.The method of claim 94 wherein the compound is administered nasally.100. The method of claim 94 wherein the compound is administeredsublingually.
 101. The method of claim 94 wherein the compound isadministered in a dosage of from about 0.01 μg/day to about 1000 μg/day.102. The method of claim 94 wherein the compound is2β-methyl-19,26,27-trinor -(20S)-1α-hydroxyvitamin D₃ having theformula:


103. A method of treating an autoimmune disease selected from the groupconsisting of multiple sclerosis, lupus, diabetes mellitus, host versusgraft rejection, and rejection of organ transplants, comprisingadministering to a subject with said disease an effective amount of acompound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 104. The method of claim103 wherein the compound is administered orally.
 105. The method ofclaim 103 wherein the compound is administered parenterally.
 106. Themethod of claim 103 wherein the compound is administered transdermally.107. The method of claim 103 wherein the compound is administeredrectally.
 108. The method of claim 103 wherein the compound isadministered nasally.
 109. The method of claim 103 wherein the compoundis administered sublingually. 110 The method of claim 103 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 111. The method of claim 103 wherein the compound is2β-methyl-19,26,27-trinor -(20S)-1α-hydroxyvitamin D₃ having theformula:


112. A method of treating an inflammatory disease selected from thegroup consisting of rheumatoid arthritis, asthma, and inflammatory boweldiseases, comprising administering to a subject with said disease aneffective amount of a compound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 113. The method of claim112 wherein the compound is administered orally.
 114. The method ofclaim 112 wherein the compound is administered parenterally.
 115. Themethod of claim 112 wherein the compound is administered transdermally.116. The method of claim 112 wherein the compound is administeredrectally.
 117. The method of claim 112 wherein the compound isadministered nasally.
 118. The method of claim 112 wherein the compoundis administered sublingually.
 119. The method of claim 112 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 120. The method of claim 112 wherein the compound is2β-methyl-19,26,27-trinor -(20S)-1α-hydroxyvitamin D₃ having theformula:


121. A method of treating a skin condition selected from the groupconsisting of wrinkles, lack of adequate skin firmness, lack of adequatedermal hydration and insufficient sebum secretion which comprisesadministering to a subject with said skin condition an effective amountof a compound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 122. The method of claim121 wherein the compound is administered orally.
 123. The method ofclaim 121 wherein the compound is administered parenterally.
 124. Themethod of claim 121 wherein the compound is administered transdermally.125. The method of claim 121 wherein the compound is administeredtopically.
 126. The method of claim 121 wherein the compound isadministered rectally.
 127. The method of claim 121 wherein the compoundis administered nasally.
 128. The method of claim 121 wherein thecompound is administered sublingually.
 129. The method of claim 121wherein the compound is administered in a dosage of from about 0.0μg/day to about 1000 μg/day.
 130. The method of claim 121 wherein thecompound is 2β-methyl-19,26,27-trinor -(20S)-1α-hydroxyvitamin D₃ havingthe formula:


131. A method of treating renal osteodystrophy comprising administeringto a subject with renal osteodystrophy an effective amount of a compoundhaving the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 132. The method of claim131 wherein the compound is administered orally.
 133. The method ofclaim 131 wherein the compound is administered parenterally.
 134. Themethod of claim 131 wherein the compound is administered transdermally.135. The method of claim 131 wherein the compound is administeredrectally.
 136. The method of claim 131 wherein the compound isadministered nasally.
 137. The method of claim 131 wherein the compoundis administered sublingually.
 138. The method of claim 131 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1 000 μg/day.
 139. The method of claim 131 wherein the compound is2β-methyl-19,26,27-trinor -(20S)-1α-hydroxyvitamin D₃ having theformula: HO OH


140. A method of treating or preventing obesity of an animal, inhibitingadipoctye differentiation, inhibiting SCD-1 gene transcription, and/orreducing body fat in an animal comprising administering to an animal inneed thereof an effective amount of a compound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 141. The method of claim140 wherein the compound is administered orally.
 142. The method ofclaim 140 wherein the compound is administered parenterally.
 143. Themethod of claim 140 wherein the compound is administered transdermally.144. The method of claim 140 wherein the compound is administeredrectally.
 145. The method of claim 140 wherein the compound isadministered nasally.
 146. The method of claim 140 wherein the compoundis administered sublingually.
 147. The method of claim 140 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 148. The method of claim 140 wherein the compound is2β-methyl-19,26,27-trinor -(20S)-1α-hydroxyvitamin D₃ having theformula:


149. The method of claim 140 wherein the animal is a human.
 150. Themethod of claim 140 wherein the animal is a domestic animal.
 151. Themethod of claim 140 wherein the animal is an agricultural animal.